CN101641114A - The treatment of inflammation and/or endotoxin shock - Google Patents

Abstract

The invention provides one or more peptides derived from the proteic C-end of Kai Morui, or their analog or derivant are used for the treatment of inflammation and/or endotoxin shock in the subject and/or handle wound in the subject and/or reduce the application of inflammatory chemokine level in the subject, and derived from one or more peptides of the proteic C-end of Kai Morui, or their analog or derivant are used for the treatment of inflammation and/or endotoxin shock, and/or treatment of wounds, or be used to reduce the application of the level of inflammatory mediator.

Description

The treatment of inflammation and/or endotoxin shock

Technical field

The present invention relates to the reduction of the level of the treatment of inflammation and/or endotoxin shock and/or inflammatory chemokine, and relate to the treatment that is used for inflammation and/or endotoxin shock, or be used for the compositions that the level of inflammatory mediator reduces.

Background technology

Inflammation is the ingredient of the pathogenesis of many human and animal's diseases, and as the result of the mankind or the systemic physics of animal, chemistry or traumatic injury and take place.Usually, immunne response causes the whole body of endogenous chemical mediator to discharge, and this can cause vasodilation, neutrophilic leukocyte migration, chemotaxis, reach the permeability increase of vascular.No matter where immunne response occurs in and what causes that its reason is identical basically by.Reply or can be chronic (continuing) for acute (of short duration).

Endotoxin shock is also sometimes referred to as septic shock, is considered to be replied by the inflammation sample owing to being exposed in the blood vessel that a large amount of endotoxins of causing take place.Be exposed to endotoxin and cause producing a large amount of cytokines, comprise TNF α and IL-1.The complement system and the cascade (coagulation cascade) of condensing comprise that factor VII also is upset.The result of this reaction can be tissue injury, heating, vasodilation, tachycardia and IC.

Inflammatory response is normally useful, and the nutrient, oxygen, antibody and the curative drug that make inflammation part enter increase, and fibrinous formation increase and toxin dilution.Yet,, can cause the damage of tissue if inflammation is undesirable or prolongation.Under these circumstances, use the anti-inflammatory medicine usually.The anti-inflammatory medicine that has two kinds of main types, corticosteroids and NSAID (non-steroidal anti-inflammatory drug) (NSAIDs).Great majority in these medicines have undesirable side effect.It is relevant with the serious side effects of the hypercortisolism (Cushing ' s disease) of mimicry that the 17-hydroxy-11-dehydrocorticosterone that prolongs gives usually, and hypercortisolism is a kind of suprarenalism that is caused by hydrocortisone generation surplus.Other possible side effect comprise weight increase, chest, face, cervical region and last back lipidosis, edema, hypertension, diabetes, poor wound healing, infect easily, thinning of skin, emotional instability and depression.The serious adverse of NSAIDS is renal failure, liver failure, ulcer and damage or postoperative continuous bleeding.Some individualities are to NSAIDs allergy, and there is high risk in the people who suffers from asthma to the severe allergic reaction of aspirin.Therefore, need to identify optional medicament with anti-inflammatory effect.

Kai Morui (Chemerin, a kind of adipose cell factor is the endogenic ligand of the plain receptor chemerin of lonely G albumen coupling R) be a kind of Abundant protein in the human inflammatory exudate (comprising ascites and synovial fluid) (Wittamer V et al.J Exp Med.2003 October 6 of being present in; 198 (7): 977-985; Meder Wet et al.FEBS Lett.2003 December 18 days; 555 (3): 495-499).Human Kai Morui (Chemerin) is as 163 aminoacid (aa) precursor (mice (Musmusculus) that is called precursor Kai Morui (ProChemerin), the muroid equivalent is 162aa) and it is excretory, the terminal truncate of its experience N-and C-is with the chemotactic protein (being 140aa in the mice) that produces 137aa (Wittamer V et al.J Exp Med.2003 October 6; 198 (7): 977-985; Zabel BA et al.J BiolChem.2005 October 14; 280 (41): 34661-34666; Wittamer V et al.JImmunol.2005 July 1; 175 (1): 487-493; Samson M et al.Eur JImmunol.1998 May; 28 (5): 1689-1700).The predict of Kai Morui has shown the structural similarity with chemotactic factor, it is described to " opposite " chemotactic factor, has unordered carboxyl-end, α-pleated sheet and β-spiral amino-end structure territory (Zabel BA et al.Exp Hematol.2006 August potentially; 34 (8): 1021-1032).This structure associates the people to be present in the cystatin (cystatin) in cathepsin and the kininogen folding, and it also experiences the Proteolytic enzyme process to realize activation (Zabel BAet al.Exp Hematol.2006 August; 34 (8): 1106-1114; Colman RW, BiolChem.2001 January; 382 (1): 65-70; Yamasaki K et al.FASEB is on October 1,2006 J.2006; 20 (12): 2068-2080).

Summary of the invention

According to first aspect, the invention provides one or more peptides, or their analog or derivant are used for the treatment of application in the medicine of inflammation in preparation derived from the proteic C-end of Kai Morui (Chemerin).

According on the other hand, the invention provides one or more peptides, or their analog or derivant are used for preparing the application of the medicine of endotoxin shock in preparation derived from the proteic C-end of Kai Morui.

According on the other hand, the invention provides one or more peptides, or their analog or derivant are used for reducing the application of the medicine of one or more inflammatory mediator levels in preparation derived from the proteic C-end of Kai Morui.

According to another aspect, the invention provides one or more peptides, or their analog or derivant, to be used for the treatment of inflammation and/or treatment endotoxin shock and/or to reduce the level of one or more inflammatory mediators derived from the proteic C-end of Kai Morui.

These one or more inflammatory mediators can comprise cytokine, chemotactic factor and the lipid of transmitting inflammation.This inflammatory mediator can comprise being selected from and comprises one or more chemotactic factors in the group of being made up of following material: TNF α, IL-1 α, IL-1 β, IL-6, IL-12, G-CSF, MCP-2 (CCL8), GRO α (CXCL 1), GRO β (CXCL2), IL-8 (CXCL8), TECK (CCL25), MCP-1 (CCL2), interferon gamma and RANTES (CCL5).Preferably, this medicine can reduce the level of TNF α.

Beat allly be, have anti-inflammatory property, can be used for the treatment of, prevent or improve inflammation and/or endotoxin shock derived from the peptide of the proteic C-end of Kai Morui.

This medicine can have the application for the treatment of and/or preventing.

Preferably, this peptide arrives between about 30 aminoacid about 5.More preferably, described peptide arrives between about 25 aminoacid about 5, and preferably, described peptide arrives between about 20 aminoacid about 5.

Preferably, this peptide comprises about 5 to about 30 aminoacid derived from the proteic C-end of Chemierin, or their analog or derivant.More preferably, described peptide arrives between about 25 aminoacid about 5, and preferably, described peptide arrives between about 20 aminoacid about 5.

Kai Morui (Chemerin) albumen is meant the processing form of Kai Morui, wherein the-terminal amino acid that exists in the precursor of precursor Kai Morui (PreProChemerin) is removed by Proteolytic enzyme, the C-end amino acid that exists in precursor Kai Morui (ProChemerin) precursor is removed by Proteolytic enzyme, thereby produces the proteic active clipped form that is called Kai Morui.

Preferably, this peptide is conigenous the Kai Morui of human form or inhuman form.Preferably, this peptide is from the Kai Morui of the mankind or mammal form.The inhuman Kai Morui of mammal can be from rodent, as rat or mice, horse, Canis familiaris L., cat, cattle, sheep or pig.

Preferably, have at least 30% or higher homogeneity derived from the peptide of the proteic C-end of Kai Morui and the peptide in the natural Kai Morui of being present in PROTEIN C-end.Preferably, described peptide and the proteic C-terminal peptide of naturally occurring Kai Morui sequence have at least 40%, 50%, 60%, 70%, 80%, 90% or higher homogeneity.

Preferably, this peptide and natural last approximately 5 to last approximately 30 of being present in Kai Morui PROTEIN C-end, preferably last approximately 10 have at least 30% or higher sequence homogeneity to last approximately 25 aminoacid.Preferably, this peptide and natural last approximately 5 to last approximately 30 of being present in Kai Morui PROTEIN C-end, preferably last approximately 10 have at least about 40%, 50%, 60%, 70%, 80%, 90% or higher sequence homogeneity to last approximately 25 aminoacid.

Preferably, 5 to 25 aminoacid in last 30 aminoacid in this peptide and the natural Kai Morui of the being present in PROTEIN C-end have at least 30% or higher sequence homogeneity.Preferably, 5 to 25 aminoacid in last 30 aminoacid of this peptide and the natural Kai Morui of being present in PROTEIN C-end have at least about 40%, 50%, 60%, 70%, 80%, 90% or higher sequence homogeneity.

Be meant the aminoacid of this PROTEIN C-end about " the last aminoacid " in the Kai Morui albumen.

Provided the full length sequence of human and Mus Kai Morui, precursor Kai Morui (ProChemerin) and precursor Kai Morui precursor (PreProChemerin) among Fig. 2 A, reflected respectively for the human protein among the Seq ID no:31,32,33, and reflected murine protein matter respectively among the Seq ID no:34,35,36.Preferably, the Kai Morui peptide has the sequence of Seq ID No:31 or 34.From other species, can easily can easily obtain from GenBank acquisition and those skilled in the art as the proteic sequence of the Kai Morui of cattle and rat.

Preferably, last 5 of the Kai Morui of this peptide and Seq ID no:31 (| human sequence) and Seq ID no:34 (mice sequence) has at least 30% or higher to last 30 aminoacid, and more preferably 40%, 50%, 60%, 70%, 80%, 90% or higher sequence homogeneity.

The percentage ratio amino acid sequence identity is defined in aligned sequences and introduces room (gap) where necessary with after the percentage ratio sequence homogeneity that obtains maximum, the percentage ratio of the amino acid residue that the aminoacid with in the naturally occurring Kai Morui albumen in the sequence is identical.Be used for determining that the comparison (Alignment) of percentage ratio sequence homogeneity can realize well known to a person skilled in the art many modes, comprise, for example, utilize BLAST and ALIGN algorithm.

This peptide can comprise interpolation, insertion, disappearance, inversion or the transposition with respect to the native sequences of Kai Morui PROTEIN C-end, so that this peptide is compared with the peptide with native sequences, has at least 50% anti-inflammatory activity, and/or at least 50% antiendotoxin shock is active, and/or one or more inflammatory mediator levels are reduced at least 50% ability.

Term " analog " or " derivant " are meant such peptide, it has the sequence different with naturally occurring sequence, but with having the natural peptide that has a sequence when observing, it comprises substantially the same or more, and at least about 50%, preferred about anti-inflammatory activity of 60%, 70%, 80% or the 90% and/or antiendotoxin shock is active, inflammatory mediator reduces active.

Peptide analogues or derivant can have any amino acid residue, comprise one or more disappearances, insertion or the modification of N or C-terminal residue.This peptide can be acetylation, acidylate, alkylation, glycosylation etc.This peptide can also comprise other aminoacid at C or N-terminal or C and N-terminal.

This peptide, analog or derivant can be the part of fusion rotein.

Compare with naturally occurring aminoacid sequence, this peptide can comprise one or more conservative amino acid replacements.

Preferably, one or more described peptides comprise and are selected from the sequence that comprises following sequence set:

PHGYFLPGQFA (Chemerin11-mice; C11m; Seq ID No:1);

PHGYFLPGQFAF (Chemerin12-mice; C12m; Seq ID No:2);

PHGYFLPGQFAFS (Chemerin13-mice; C13m; Seq ID No:3);

AGEDPHGYFLPGQFA (Chemerin15-mice; C 15m; Seq ID No:4);

AGEDPHGYFLPGQFAF (Chemerin16-mice; C16m; Seq ID No:5);

AGEDPHGYFLPGQFAFS (Chemerin17-mice; C17m; Seq ID No:6);

DPHGYFLPGQFA (Chemerin12A-mice; C12Am; Seq ID No:7);

EDPHGYFLPGQFA (Chemerin13A-mice; C13Am; Seq ID No:8);

GEDPHGYFLPGQFA (Chemerin14A-mice; C14Am; Seq ID No:9);

DPHGYFLPGQFAF (Chemerin13B-mice; C13Bm; Seq ID No:10);

EDPHGYFLPGQFAF (Chemerin14B-mice; C14Bm; Seq ID No:11);

GEDPHGYFLPGQFAF (Chemerin15A-mice; C 15Am; Seq ID No:12);

DPHGYFLPGQFAFS (Chemerin14C-mice; C14Cm; Seq ID No:13);

EDPHGYFLPGQFAFS (Chemerin15B-mice; C15Bm; Seq ID No:14);

GEDPHGYFLPGQFAFS (Chemerin16A-mice; C16Am; Seq ID No:15);

PHSFYFPGQFA (the Chemerin11-mankind; C11h; Seq ID No:16);

PHSFYFPGQFAF (the Chemerin12-mankind; C12h; Seq ID No:17);

PHSFYFPGQFAFS (the Chemerin13-mankind; C13h; Seq ID No:18);

AGEDPHSFYFPGQFA (the Chemerin15-mankind; C15h; Seq ID No:19);

AGEDPHSFYFPGQFAF (the Chemerin 16-mankind; C 16h; Seq ID No:20);

AGEDPHSFYFPGQFAFS (the Chemerin 17-mankind; C 17h; Seq ID No:21);

DPHSFYFPGQFA (the Chemerin12A-mankind; C12Ah; Seq ID No:22);

EDPHSFYFPGQFA (the Chemerin13A-mankind; C 13Ah; Seq ID No:23);

GEDPHSFYFPGQFA (the Chemerin14A-mankind; C14Ah; Seq ID No:24);

DPHSFYFPGQFAF (the Chemerin13B-mankind; C13Bh; Seq ID No:25);

EDPHSFYFPGQFAF (the Chemerin14B-mankind; C14Bh; Seq ID No:26);

GEDPHSFYFPGQFAF (the Chemerin 15A-mankind; C 15Ah; Seq ID No:27);

DPHSFYFPGQFAFS (the Chemerin 14C-mankind; C 14Ch; Seq ID No:28);

EDPHSFYFPGQFAFS (the Chemerin15B-mankind; C 15Bh; Seq ID No:29); And

GEDPHSFYFPGQFAFS (the Chemerin16A-mankind; C16Ah; Seq ID No:30) or their analog or derivant.

Preferably, one or more these peptides comprise the sequence that is selected from the group that comprises following sequence:

AQAGEDPHGYFLPGQFAFS (Chemerin19-mice; C19m; Seq IDNo:37); And

QRAGEDPHSFYFPGQFAFS (the Chemerin19-mankind; C 19h; Seq IDNo:38); Or their analog or derivant.

Preferably, this peptide with above be called as Seq ID No:1, one or more peptides of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 and have at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher sequence homogeneity.

Preferably, this peptide and one or more peptides that above are called as Seq ID No:37 or 38 have at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher sequence homogeneity.

Preferably, derived from the analog of the peptide of the proteic C-end of Kai Morui or the small molecule mimetics that derivant comprises peptide.

This peptide, analog or derivant can be separated from natural system, perhaps can synthetically or be recombinantly produced.Synthetic peptide can be produced by the chemical method of standard, comprises by auto-programming synthetic.

Recombinant peptide can use with purified form.Replacedly, can use supernatant from the cell of express recombinant peptide.

This peptide, analog or derivant can form the part of bigger albumen or molecular complex.

This peptide can be straight chain or cyclic.

This peptide can comprise the main chain of resistant protease.

This peptide can be included in the modification of C and/or N-terminal.

This peptide can be labeled by methods known in the art, as radioactive label, fluorescent labeling, mass spectrum label, biotin etc.

Described medicine can comprise other active component, comprises the medicament of other known anti-inflammatory agents and/or other known antiendotoxin shock medicaments and/or other known reduction chemotactic factor levels.

Described medicine can also comprise pharmaceutical excipient.This excipient can comprise big macromole, as protein, polysaccharide, polylactic acid, polyglycolic acid, polyamino acid, amino acid copolymer, trehalose, lipid aggregation and nonactive virion.Such excipient is well known to those skilled in the art.

Described medicine can also comprise one or more in buffer agent, viscosifier, solvent, stabilizing agent and the antiseptic.

The route of administration of described medicine can for by in parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intra-arterial, the damage, intraarticular, part, per os, rectum, per nasal, suction or injection of any other suitable way or infusion.

The dosage of used peptide depends on peptide, target and processing.Dosage and approach determine it is in gengral practitioner's technical scope.The dosage regimen of normal dose can change in the scope of about 100mg/kg at about 1pg/kg, and more preferably, this dosage, more preferably changes in the scope of about 100ng/kg at about 10pg/kg to about 1mg/kg at about 10pg/kg.Preferably, these dosage are dosage every day.

Beat all is have been found that the dosage that is low to moderate 0.32ng/kg according to peptide of the present invention has usefulness to the intravital aseptic peritonitis of mice, and needs to use the dexamethasone of 1.2mg/kg just can observe the usefulness of similar degree.

Preferably, can be intended to the dosed administration to about 1mg/kg, more preferably, arrive the dosed administration that about 100ng/kg or about 10pg/kg arrive about 10ng/kg with about 10pg/kg with about 10pg/kg according to the medicine of application of the present invention.These dosage are than low at least three the logarithm levels (log) of dosage of needed dexamethasone.

According to a further aspect in the invention, provide treatment/handle, prevent or improve the method for inflammation in the subject, comprised giving the experimenter one or more peptide or their analog or derivants derived from the proteic C-end of Kai Morui.

According to a further aspect in the invention, provide treatment/handle, prevent or improve the method for endotoxin shock in the subject, comprised giving the experimenter one or more peptide or their analog or derivants derived from the proteic C-end of Kai Morui.

According to a further aspect in the invention, provide the method that reduces intravital one or more inflammatory mediator levels of experimenter, comprised giving the experimenter one or more peptide or their analog or derivants derived from the proteic C-end of Kai Morui.

Described treatment (processing) can be curative, preventative or beauty treatment usefulness.

Preferably, this peptide is given with effective dose, and effective dose is, and is enough to (i) and induces or cause inflammation to reduce, or prevent or reduce inflammation; (ii) induce or cause the minimizing of endotoxin shock, or prevent or reduce endotoxin shock; And/or (iii) reduce the amount of one or more inflammatory mediator levels.

Alternately, medicine of the present invention can directly be applied to medical apparatus to reduce the risk of the relevant device of inflammation.This can by with medicament administration in the surface of this device or by realizing with one or more surfaces of flooding described device derived from the peptide of the proteic C-end of Kai Morui or their analog or derivant.

According on the other hand, the invention provides with one or more derived from the peptide of the proteic C-end of Kai Morui or the medical apparatus of their analog or derivant dipping.

This medical apparatus can be support or conduit.

According to another aspect, the invention provides with one or more derived from the peptide of the proteic C-end of Kai Morui or the wound dressing or the binder of their analog or derivant dipping.

The related inflammation in any aspect of the present invention can be relevant with following disease, for example juvenile chronic arthritis, SpA, systemic sclerosis (scleroderma), constitutional inflammatory myopathy (dermatomyositis, polymyositis), sjogren syndrome (Sjogren ' s syndrome), systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia (immunopancytopenia, paroxysmal nocturnal hemoglobinuria), AT (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Graves' disease, struma lymphomatosa, teenager lymphocyte thyroiditis, atrophic thyroiditis), the autoimmunity inflammatory diseases is (as allergic encephalitis, multiple sclerosis, insulin-dependent diabetes, the autoimmunity retinitis, thyrotoxicosis, scleroderma, systemic lupus erythematosus (sle), rheumatoid arthritis, inflammatory bowel is (as Crohn disease, ulcerative colitis, regional enteritis, distal ileitis, granulomatous enteritis, segmental enteritis, distal ileitis), autoimmune thyroid disease, pernicious anemia, allograft rejection, diabetes, immune-mediated nephropathy (glomerulonephritis, the renal tubules interstitial nephritis), the demyelinating disease of maincenter and peripheral nervous system such as multiple sclerosis, primary demyelination polyneuropathy or Guillain Barre syndrome, and chronic inflammatory demyelinative polyneuropathy, liver-gallbladder disease such as infectious hepatitis (first, second, third, fourth, hepatitis E and other are non-has a liking for hepatovirus), the autoimmunity chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, sclerosing cholangitis, glutelin sensitivity enteropathy, Whipple, autoimmunity or immune-mediated dermatosis comprise bullous dermatosis, erythema multiforme and contact dermatitis, psoriasis, allergosis such as asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity and urticaria, pulmonary's immune disease such as eosinophilic pneumonia, congenital pulmonary fibrosis and hypersensitivity pneumonitis (hypersensitivitypneumonitis), transplant relevant disease and comprise transplant rejection and graft versus host disease, infectious disease comprises viral disease such as AIDS (HIV infection), herpes etc., bacterial infection, fungal infection, protozoal infections, parasitic infection, and respiratory syncytial virus, HIV (human immunodeficiency virus) etc., eczema and endotoxin shock.

According on the other hand, the invention provides the peptide that can treat, prevent or improve inflammation, this peptide be selected from comprise have Seq ID No:1, group that the peptide of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 sequence and their analog or derivant are formed.

According on the other hand, the invention provides the peptide that can treat, prevent or improve inflammation, this peptide be selected from comprise have Seq ID No:37, group that the peptide of 38 sequence and their analog or derivant are formed.

According on the other hand, the invention provides the peptide that can treat, prevent or improve endotoxin shock, this peptide be selected from comprise have Seq ID No:1, group that the peptide of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 sequence and their analog or derivant are formed.

According on the other hand, the invention provides the peptide that can treat, prevent or improve endotoxin shock, this peptide be selected from comprise have Seq ID No:37, group that the peptide of 38 sequence and their analog or derivant are formed.

According on the other hand, the invention provides the peptide that can reduce one or more inflammatory mediator levels, this peptide be selected from comprise have Seq ID No:1, group that the peptide of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 sequence and their analog or derivant are formed.

According on the other hand, the invention provides the peptide that can reduce one or more inflammatory mediator levels, this peptide be selected from comprise have Seq ID No:37, group that the peptide of 38 sequence and their analog or derivant are formed.

According on the other hand, the invention provides the pharmaceutical composition that comprises one or more peptides, this peptide be selected from comprise have Seq ID No:1, group that the peptide of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 and 30 sequence and their analog or derivant are formed.

According on the other hand, the invention provides and comprise pharmaceutical compositions a kind of or a plurality of kinds, this peptide is selected from the group that the peptide that comprises the sequence with Seq ID No:37 and 38 and their analog or derivant are formed.

This pharmaceutical composition can be used for the treatment of and/or prevention of inflammation and/or treat and/or prevent endotoxin shock and/or be used to reduce the level of one or more inflammatory mediators (as cytokine and chemotactic factor).

According to another aspect, the invention provides have Seq ID No:1, peptide or their analog or the derivant of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 sequence.

According on the other hand, the invention provides peptide or their analog or the derivant of sequence with Seq ID No:37 or 38.

According to another aspect, the invention provides the peptide that has at least 50% sequence homogeneity with peptide with Seq ID No:1, sequence of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30.Preferably, this peptide has at least 60%, 70%, 80%, 90% or 95% sequence homogeneity with the peptide with Seq ID No:1, sequence of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30.

According to another aspect, the invention provides the peptide that has at least 50% sequence homogeneity with the peptide with sequence of Seq ID No:37 or 38.Preferably, this peptide has at least 60%, 70%, 80%, 90% or 95% sequence homogeneity with the peptide with sequence of Seq IDNo:37 or 38.

According on the other hand, the invention provides one or more peptides, or their analog or derivant are used for handling the application of the medicine of wound in preparation derived from the proteic C-end of Kai Morui.

According on the other hand, the invention provides treatment, prevent or improve the method for experimenter's wound, comprise giving the experimenter one or more peptides derived from the proteic C-end of Kai Morui, or their analog or derivant.

According on the other hand, the invention provides the pharmaceutical composition that comprises one or more peptides that is used to handle wound, this peptide be selected from comprise have Seq ID No:1, group that the peptide of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,37 or 38 sequence or their analog or derivant are formed.

According on the other hand, the invention provides one or more peptides derived from the proteic C-end of Kai Morui, or their analog or derivant, to be used for treating and/or preventing and/or the level that treats and/or prevents and/or be used to reduce one or more inflammatory mediators (as cytokine and chemotactic factor) of endotoxin shock of inflammation.

According on the other hand, the invention provides one or more peptides derived from the proteic C-end of Kai Morui, or their analog or derivant, to be used for the processing of wound.

Those skilled in the art will recognize that any preferable feature that above discloses can be applied to any aspect of the present invention.

Description of drawings

Below will only with reference to the following drawings and embodiment preferred implementation of the present invention be described by way of example.

Figure 1A to Fig. 1 F-Figure 1A and Figure 1B show Chemerin140 and suppress generation by the inflammatory mediator of macrophage in the dependent mode of Proteolytic enzyme.Utilize Luminex and ELISA analysis of experiments cytokine-expressing from the supernatant of macrophage or activatory macrophage (handling) with 100ng/mlLPS and 20ng/ml interferon gamma.Shown in the recombinant Mus Kai Morui (Chemerin) of dosage or dexamethasone exist or situation about lacking under and protease inhibitor leupeptin (leupeptin) (15mg/ml) exist or situation about lacking under incubated cell.Fig. 1 C-by qRT-PCR (IL-10, TGF β) with the horizontal quantification of macrophage cytokines mRNA be standardized as HPRT.Fig. 1 D-is at LPS/IFN γ-usefulness Kai Morui (0.1-1pM) ± pertussis toxin, PT (PTX before exciting; 200ng/ml) PM Φ is carried out pretreatment.Fig. 1 E-, stimulated 4 hours, 8 hours or 15 hours with LPS/IFN γ 1 hour ± PTX of PM Φ pretreatment subsequently with Kai Morui (1pM).With respect to the sample that LPS/IFN γ handles, * * *, p＜0.001; *, p＜0.01; *, p＜0.05.With respect to the sample that Kai Morui handles, ###, p＜0.001; ##, p＜0.01; #, p＜0.05.Fig. 1 F-to peritoneal macrophages (PM Φ) pretreatment 1h, uses LPS (100ng/ml) and IFN γ (20ng/ml) stimulation 15 hours with Kai Morui (1pM), Kai Morui (1pM)+protease inhibitor (leupeptin [Leu], E-64, Pefabloc[Pef], Pepstatin A [Pep A], calpain inhibitor (Calpeptin) [Cal], cathepsin S inhibitor [Cath S], cathepsin L inhibitor [Cath L]) subsequently.Chart shows the meansigma methods ± SEM from 3-8 independent experiment.Nd: be lower than the detectability of this test, ns is not remarkable;

Fig. 2 A-shows the aminoacid sequence comparison of mankind's (top sequence) and mice (below sequence) Kai Morui (Tig2).Utilize PeptideCutter (http://www.expasy.org/tools/peptidecutter/) that the aminoacid sequence of human (the Protein Data Bank accession number is NP_002880) and Mus (NP_082128) Kai Morui is compared and analyzes the trypsin cracking site of predicting with generation (dark vertical line).Runic and gray full length sequence are the precursor (sequence of PreProChemerin) (human sequence is Seq ID No:33, and the mice sequence is Seq ID No:36) of precursor Kai Morui.The removed sequence of Lycoperdon polymorphum Vitt N-terminal aminoacid is precursor Kai Morui (ProChemerin) sequence (human sequence is Seq ID No:32, and the mice sequence is Seq ID No:35).The sequence that the removed runic sequence of Lycoperdon polymorphum Vitt N-terminal and C-end amino acid is Kai Morui (human sequence is SeqID No:31, and the mice sequence is Seq ID No:34).

Give the sequence of C-terminal peptide C11m (Seq ID No:1), C13m (Seq ID No:2), C15m (Seq ID No:3) and C 17m (Seq ID No:4);

Fig. 2 B-shows the peptide derived from the C-end portion of Kai Morui of preceding-inflammatory mediator that inhibition produces by activated macrophage.In this width of cloth figure, label peptide C13, C15 and C17 refer to the peptide that is described as C13m, C15m and C17m in the preamble respectively;

Fig. 3-show Kai Morui peptide of comparing the macrophage chemotactic performance that demonstrates seldom with Chemerin140.In this width of cloth figure, label peptide C11, C13, C15 and C17 refer to the peptide that is described as C11m, C13m, C15m and C17m in the preamble respectively;

Fig. 4 A to Fig. 4 H-shows the chemotaxis of the supernatant mediation of being handled by Kai Morui (Chemerin), Kai Morui peptide and Kai Morui.Make process or do not pass through pertussis toxin, PT pretreatment (PTX; 200ng/ml) 30 minutes PM Φ (0.4 * 10 ⁶) chemoattractant migration in the base apertures of Boyden chamber of improvement surpasses 4 hours (Fig. 4 A; RmChemerin, Fig. 4 B; C15, Fig. 4 C; C11, Fig. 4 D; C13, Fig. 4 E; C19, Fig. 4 F; C6, Fig. 4 G; C8).Fig. 4 H-makes PM Φ (7.5 * 10 ⁵) the conditioned medium of the macrophage of handling from untreated macrophage with LPS/IFN γ ± Kai Morui or C15 in the base apertures of Boyden chamber of improvement moves above 4 hours.Chart shows the average Migration Index ± SEM (n=4 independently experiment) of each processed group.With respect to PM Φ+PTX, * * *, P＜0.001; *, P＜0.01; *, P＜0.05 (Xue Shengshi t check).Reference marks peptide C11, C13, C15 and C19 among this figure are meant the peptide that is described as C11m, C13m, C15m and C19m in the preamble respectively;

Fig. 5-show macrophage demonstrates towards the chemotaxis from the conditioned medium of the macrophage of Kai Morui-processing and reduces.In the figure, reference marks peptide C15 and C17 are meant the peptide that is described as C15m and C17m in the preamble respectively;

Fig. 6-showing the Chemerin15-mice suppresses zymosan-inductive peritonitis.Reference marks peptide C15 among this figure is meant the peptide that is described as C15m in the preamble.Z is meant zymosan;

Fig. 7 A to Fig. 7 G-shows chemerin15 and has improved zymosan in the mice-inductive peritonitis.Fig. 7 A and Fig. 7 B-C57B 16/J mice are injected PBS or zymosan (10 μ g, each chamber～2 * 10 again by i.p. administration PBS or chemerin15 (0.32ng/kg) after 1 hour ⁶Individual granule).At a plurality of time points (Fig. 7 A and Fig. 7 B; 5-6 mice/processing) (Fig. 7 C-Fig. 7 E or after 4 hours; 6-15 mice/group) collect the peritoneal effusion cell by peritoneal lavage.Fig. 7 C to Fig. 7 E-is the total cell number quantification in the irrigating solution, and utilizes facs analysis to determine that cell forms (neutrophilic leukocyte and mononuclear phagocyte).Cell resists-Fc γ RII/III blocking-up with 2.4G2, and dyes with Ly-6G-PE and 7/4-FITC.Make up door (gate) around two groups, these two groups are neutrophilic leukocyte (N; 7/4 height, the Ly-6G height) and inflammatory mononuclear cell (Mo; 7/4 height, Ly-6G is low).Fig. 7 E-shows each processed group behind zymosan 4 hours typical FACS figure.Fig. 7 F-passes through the TNF α and the KC of elisa assay peritoneal lavage, and by Luminex analysis of experiments IL-6, IL-1 β and MCP-1.C15; Chemerin15, Z; Zymosan.For the animal of zymosan-processing, * * *, P＜0.001; *, P＜0.01** (Xue Shengshi t check).Fig. 7 G-in advance 1 hour (C 15 pretreatment) or after 2 hours (C15 post processing) give zymosan (10 μ g) and C15 (8Pg) or PBS to mice (6-8/ processing) i.p..Peritoneal lavage excites back 4h to carry out at zymosan.Reference marks peptide C15 among this figure is meant the peptide that is described as C15m in the preamble;

Fig. 8 A-Fig. 8 D-shows anti--Kai Morui antibody Zhong with Kai Morui material and peritonitis is worsened.Fig. 8 A-PM Φ is used for macrophage chemotaxis test (seeing Fig. 7 for details carries out), and make its existence or do not have anti--rmChemerin antibody or the situation of contrast IgG under towards RANTES, Kai Morui or C15 migration.Chart shows the average Migration Index ± SEM (n=4 independently experiment) for each processed group.With respect to chemoattractant, * * *, P＜0.001; *, P＜0.01; *, P＜0.05.Fig. 8 B-is under the situation that has or do not exist anti--rmChemerin antibody or contrast IgG, and PM Φ reaches 1 hour with 1pM C15 or 1pM Kai Morui pretreatment, uses LPS (100ng/ml) and IFN γ (20ng/ml) to stimulate subsequently 15 hours.Measure the average expression ± SEM of the RANTES in the macrophage supernatant after 16 hours by ELISA (n=4 independently experiment).With respect to the sample of LPS/IFN γ-processing, * *, P＜0.01; *, P＜0.05.Fig. 8 C and Fig. 8 D-C57B16/J mice i.p. give PBS, anti--rmChemerin antibody (100ng/ mice) or contrast IgG (100ng/ mice), injection PBS or zymosan (10 μ g/ chamber) after 1 hour.Inject back 4 hours and 24 hours at zymosan and collect the peritoneal effusion cell, and handle as shown in Figure 7 by peritoneal lavage.Z; Zymosan, ChAB; Anti--rmChemerin antibody.With respect to the mice * * of zymosan-excite, P＜0.01.Reference marks peptide C15 among this figure is meant the peptide that is described as C15m in the preamble;

Fig. 9-show only injects 0.32ng/kg C15m and can not induce neutrophilic leukocyte or macrophage to raise, but can reduce peritoneum TNF alpha levels.Reference marks peptide C15 among this figure is meant the peptide that is described as C15m in the preamble;

The human peptide of the Chemerin13-of Figure 10-show modification suppresses that RANTES and TNF alpha transcriptional thing express in the mouse macrophage.Reference marks peptide hC13 among this figure is meant the C13h peptide of modification;

Figure 11-show C17m does not influence the inductive macrophage chemotaxis of C140-.Reference marks peptide C17 among this figure is meant the peptide that is described as C17m in the preamble;

Figure 12-show Chemerin15-mice and Chemerin17-mice suppress the TNF α secretion of the mouse macrophage that stimulated by zymosan.Reference marks peptide C15 and C17 among this figure are meant the peptide that is described as C15m and C17m in the preamble respectively.Dexa is meant dexamethasone;

Figure 13-be the chessboard analysis, show Chemerin140 and Chemerin15-mice and induce real macrophage chemotaxis rather than chemokinesis.Reference marks peptide C15 among this figure is meant the peptide that is described as C15m in the preamble;

Figure 14 A-Figure 14 B-shows chemerin15 and suppress the scope that mononuclear cell and neutrophil recruitment surpass C15 and zymosan dosage in zymosan peritonitis.Figure 14 A-C57B16/J mice (5-6 animal/processing) is given PBS or C 15 (0.32ng/kg) by i.p, injection PBS or zymosan dosage range (10 μ g-1mg after 1 hour; A).Figure 14 B-mice (5-6 animal/processing) i.p give PBS or C 15 dosage ranges (4-40pg/ mice), injection PBS or zymosan (10 μ g behind the 1h; 2 * 10 ⁶Individual granule/chamber).Zymosan excites back 4 hours and collects the peritoneal effusion cell by peritoneal lavage.Described in Fig. 7,, and utilize facs analysis to determine cell composition (neutrophilic leukocyte and mononuclear phagocyte) with the total cell number quantification in the irrigating solution.C15; Chemerin15, Z; Zymosan.With respect to the animal that zymosan is handled, * * *, P＜0.001; *, P＜0.01**; P＜0.05* (Xue Shengshi t check).Reference marks peptide C15 or chemerin15 among this figure are meant the peptide that is described as C15m in the preamble; And

Figure 15-show the fluoremetry by the zymosan identification of macrophage, it is expressed as relative discrimination index.Experiment is in the existence of various concentration or lack under the situation of C15 and carry out.Average data represented four set, standardized experiment (± s.e.m.).

The specific embodiment

Reference marks Chemerin140 herein or C140 are meant the amino acid whose mice Kai Morui of 140 of serial ID No:34 (Chemerin) albumen (Chemerin-140-mice).

Embodiment

Chemerin140 applies antiinflammatory action to the activated macrophage of being eliminated (abrogated) by protease inhibitor

Previous research has shown that the C-of the serine protease cracked precursor Kai Morui (ProChemerin) that is discharged by polymorphonuclear cell (PMN) is terminal and has discharged its chemotactic potential (Wittamer V et al.J Immunol.2005 July 1 after threshing; 175 (1): 487-493).Yet the antiinflammatory action of the peptide that further proteolytic treatment produced by Kai Morui is to possess novelty and creationary.

Under multiple condition, cultivate Mus peritoneal macrophages (PM θ is also referred to as PM Φ herein): be untreated; LPS (100ng/ml) and IFN γ (20ng/ml), 15 hours; Kai Morui (1pM) pretreatment 1 hour, LPS/IFN γ subsequently, 15 hours; Leupeptin (protease inhibitor; 15mg/ml) and Kai Morui (1pM), 1 hour, LPS/IFN γ subsequently, 15 hours; Or dexamethasone (positive control; 1 μ M, pretreatment 1 hour, LPS/IFN γ subsequently, 15 hours.

Analysis is from the chemotactic factor content of the supernatant of the macrophage of Kai Morui+lipopolysaccharide/interferon-(LPS/IFN γ)-processing, the result shows, compare with the sample of LPS/IFN γ-processing, the cell that Kai Morui handles shows remarkable low-level TNF α (70%), IL-12p40 (54%), RANTES (CCL5; 40%), IL-6 (42%) and IL-1 β (60%) (n=5; P＜0.001, Figure 1A and Figure 1B).This antiinflammatory action is subjected to the inhibition (it prevents any antiinflammatory action when being added into macrophage) of broad-spectrum protease inhibitor (leupeptin), shows the cracked importance of Kai Morui other in the generation of these anti-inflammatory peptides (Figure 1A and Fig. 1 F).

Anti-by utilizing-the Kai Morui neutralizing antibody, what further show is that these effects are that Kai Morui is specific; Described antibody has been removed the antiinflammatory action (Fig. 8 B) of Kai Morui.

Block diagram among Figure 1A shows the average expression as the cytokine of determining by Luminex test ± SEM.For each processing, experiment is carried out with in triplicate mensuration.Show not on the same group the representative data of three independent experiments of cell of employing from C57B 16/J mice.Unless statement in addition is with respect to the sample of LPS/IFN γ-processing, p＜0.001***; P＜0.01**.Dexa is meant dexamethasone (1mM).

Figure 1B shows and the similar result about discussing among Figure 1A above, also has other data to show the effect of Kai Morui of the variable concentrations of 0.1pM, 0.5pM and 1.0pM.

In addition, Fig. 1 C shows that Kai Morui induces the expression of the mRNA that is used for anti-inflammatory cytokines TGF β (54%) and IL-10 (89%).

The effect of Kai Morui is a dose dependent, observes maximum at 1pM and replys (Figure 1B and Fig. 1 C), and to the pertussis toxin, PT sensitivity, show the link coupled GPCR of this and Gai-relevant (Fig. 1 D).

In addition, 4h, 8h and 15h observe antiinflammatory action after giving LPS/IFN γ, and are all eliminated (Fig. 1 E) by PTX at all time point antiinflammatory actions.

Previous research shows that the C-end of the serine protease cracked precursor Kai Morui (prochemerin) that granulocyte discharges also discharges its chemotactic potential (Wittamer, V., et al, (2005), J Immunol 175:487-493) after threshing.Studied the probability that Mus Kai Morui can experience the further proteolytic treatment that the enzyme that discharged by Mus M Φ activation back carries out.About what Figure 1A discussed, its antiphlogistic effects (Figure 1A and Fig. 1 F) has been eliminated in administration in the time of Kai Morui and leupeptin (a kind of serine and cystatin) as mentioned.E-64 (a kind of cystatin) also shows this effect, and acid protease inhibitor Pepstatin A (Pepstatin A) and serpin Pefabloc are for the inhibition of the activatory Kai Morui of M Φ-mediation inoperative (Fig. 1 F) simultaneously.These data show mode that Kai Morui relies on cysteine proteinase to M Φ activation play inhibitory action.Cathepsin L inhibitor (Z-FF-FMK), cathepsin S inhibitor (Z-FL-COCHO) and calpain I and II inhibitor, calpain inhibitor (calpeptin) are used to further survey the specific cysteine protease (Fig. 1 F) that relates in the Kai Morui cracking.The antiinflammatory action of discovery Kai Morui depends on calpain and cathepsin S but is independent of cathepsin L.In a word, the result shows that first the cracking of specific cysteine protease-mediation that the Mus M Φ of classical activatable can be by parent molecule is converted into the activatory effective inhibitor peptides of M Φ with Kai Morui, and most probable comprises calpain II and cathepsin S.

The terminal Kai Morui peptide of C-with anti-inflammatory activity

Utilize among the Ensembl a series of 11-20aa peptides of sequence alignment functional design as the indicant of important conserved residues (indication, indicator) and called after C11m (P144-A154; PHGYFLPGQFA Seq ID No:1), C13m (P144-S156; PHGYFLPGQFAFS Seq ID No:3), C15m (A140-A154; AGEDPHGYFLPGQFA Seq ID No:4) and C17m (A140-S156; AGEDPHGYFLPGQFAFS Seq ID No:6), C19 (A138-S156; AQAGEDPHGYFLPGQFAFS Seq ID No:37), N19 (E23-K41; ELSETQRRSLQVALEEFHK Seq ID No:44) and M20 (K86-K105; KPECTIKPNGRRRKCLACIK Seq ID No:45).Fig. 2 A shows for some the sequence alignment in these peptides.Kai Morui peptide (1pM-100nM) is characterized in the macrophage activation test according to described testing program.

Mus PM θ is cultivated under multiple condition: untreated, LPS (100ng/ml) and IFN γ (20ng/ml), 15 hours; Kai Morui peptide (concentration is 1pM-100nM) pretreatment 1h, LPS/IFN γ subsequently, 15 hours.The concentration representative that shows in two experiments for the optimum effective dose of each peptide.The average expression of histogram graph representation RANTES among Fig. 2 B and TNF α albumen ± SEM.For each processing, experiment is carried out with in triplicate mensuration.Show not on the same group the representative data of five independent experiments of cell of employing from the C57B16/J mice.Sample with respect to LPS/IFN γ processing.p＜0.01**；p＜0.001***。

C-terminal peptide C13m (100pM), C15m (1pM) and C17m (1pM) suppress LPS/IFN γ-inductive RANTES secretion (C13m-32%; C15m-41%; C17m-49%) and TNF alpha expression (C13m-10%; C15m-56%; C17m-66%, Fig. 2 B).C15m and C17m suppress macrophage activation and reach the degree that is similar to when using the C130 of same concentrations.

Similar result has been shown in the table 1, and wherein C-terminal peptide C 13 and C 19 moderates suppress LPS/IFN γ-inductive RANTES and TNF alpha expression, and optimal dose is 100pM (table 1).Yet Chemerin15 (C15) has kept by the anti-inflammatory activity shown in the proteoclastic Kai Morui and has suppressed cytokine-expressing to have similar usefulness and potential (optimal dose 1pM) with Kai Morui.In addition, C11, N-terminal peptide (N19), middle stream peptide (stage casing peptide, midstream peptide) are (M20) and control peptide (out of order C15; C15-S, GLFHDQAGPPAGYEF; Seq ID No:39 and saltant C15; C15-M, AGEDPHGYALPGQAA; Seq ID No:40) in M Φ activation test, lacks anti-inflammatory activity.Also find by solidify and precursor Kai Morui (prochemerin) burst times of the protease of fibrinolysis cascade between the 6aa (RALRTK that removes; Seq ID No:41) and 8aa (FSRALRTK; Seq ID No:42) peptide is called C6 and C8 and does not detect anti-inflammatory activity in M Φ activation test.

Table 1

With reference to table 1, as cultivating the anti-inflammatory activity-Mus PM Φ of the peptide in Kai Morui-source as described in Figure 1B, and excite (challenge) 15h with LPS (100ng/ml) and IFN γ (20ng/ml), use/without peptide (0.1pM-100nM) pretreatment 1h.When peptide showed anti-inflammatory property, the percentage ratio of LPS/1FN γ-inductive macrophage activation suppressed the effect (1pM Kai Morui and C 15 or 100pM C13 and C19) of representative with optimal dose.Peptide sequence is: C11 (P144-A154; PHGYFLPGQFA), C13 (P144-S156; PHGYFLPGQFAFS), C15 (A140-A154; AGEDPHGYFLPGQFA), C19 (A138-S156; AQAGEDPHGYFLPGQFAFS; Seq ID:No.37), N19 (E23-K41; ELSETQRRSLQVALEEFHK Seq ID No:44) and M20 (K86-K105; KPECTIKPNGRRRKCLACIK Seq ID No.45).Control peptide: out of order C15 (C15-S; GLFHDQAGPPAGYEF) and saltant C15 (C15-M; AGEDPHGYALPGQAA; F148A﹠amp; F153A).Data represented by ELISA and Luminex test determination 4-8 time independently the average percent of the cytokine that produces of the macrophage by classical activatable of experiment suppress.

Chemerin140, rather than the terminal deutero-anti-inflammatory peptides of its C-are effective macrophage chemoattractants

The macrophage chemoattractant characteristic that Boyden chamber (Boyden-chamber) test of improvement is used to show C140.Observe mice Chemerin140 and when 10nM, have best chemotactic typical bell shaped curve, descend thereafter, infer receptor desensitization or chemoattractant gradient then can take place destruction (breakdown) (Fig. 3).This is also shown in Fig. 4 A, and other data show the effect of pertussis toxin, PT pretreatment (PTX:200ng/ml) in addition.

PM θ (0.5 * 10 ⁶) chemoattractant (Chemerin140 or Kai Morui peptide) migration in the hole of the Boyden chamber bottom of improvement surpasses 4h.Filter is fixed in 4% formaldehyde, with DAPI the nucleus of migration is implemented dyeing and made it visual subsequently.Serum-free medium (SFM) is as negative control, macrophage chemoattractant RANTES (25ng/ml; 3nM) as positive control.Chart shows for the average Migration Index of each processed group (n=5-6) (chemoattractant % threshold zone/SFM% threshold zone) ± SEM.For the hole that SFM handles, p＜0.001***; P＜0.01**; P＜0.05*.

With C140 (1pM-50nM) or positive control CC chemotactic factor RANTES (25ng/ml; 3nM) compare, observe C11m, C13m and C15m (1pM-100nM) and have very little chemotactic activity.Observe maximum macrophage migration at 100pM C15m and 10nM C13m and C11m place.Yet C17m does not show chemotactic activity (0.1pM-500nM in all test concentrations; N=5 independently tests; (Fig. 3).The result is also shown in Fig. 4 B-Fig. 4 D, and other data show the effect of pertussis toxin, PT pretreatment (PTX:200ng/ml) in addition.With reference to Fig. 4 E, C 19 does not show chemotactic activity (0.1pM-500nM in all test concentrations yet; Fig. 4 E).Therefore, determined that peptide derived from Kai Morui has kept anti-inflammatory activity but do not show chemotactic activity to M Φ, this shows different function-specific component of the Kai Morui that existence can be developed by therapeutic.It is found that the peptide derived from precursor Kai Morui (prochemerin), C6 and C8 lack M Φ anti-inflammatory activity, also all can not induce M Φ migration (0.1pM-500nM in all test concentrations; Fig. 4 F-G).Therefore, as if data show that main chemotactic substance is a cracked Kai Morui molecule itself, or the peptide that does not also identify.

Show that Kai Morui and chemerin15 induce other embodiment that generally suppress by the chemical attractants deposits yields of macrophage

Set effect (Glabinski, A.R., et at., (1998) .Neuroimmunomodulation5:166-171 during the immunocyte of chemoattractant between inflammatory phase of considering M Φ-source raised; Huang, D.J.et al., (2001) .J.Exp.Med.193:713-726), untreated M Φ during the conditioned medium use is tested from chemotaxis and the M Φ that handles and only handle by Kai Morui+LPS/IFN γ, C15+LPS/IFN γ by LPS/IFN γ, with assessment by Kai Morui and synthetic C-terminal peptide to the active inhibition degree of M Φ, C15 can further influence M Φ raises (referring to Fig. 4 H).Untreated M Φ-conditioned medium itself does not show the chemotactic activity (Migration Index 1.0 ± 0.15) for M Φ; Yet the macrophage culture medium of LPS/IFN γ-processing induces that M Φ is chemotactic to significantly improve (Migration Index 9.3 ± 0.4; Fig. 4 H).In addition, M Φ shows from the macrophage of Kai Morui+LPS/IFN γ and C15+LPS/IFN γ-processing and reduces by 49% and 55% (Fig. 4 H) respectively towards the chemotaxis of conditioned medium.This shows that Kai Morui and C 15 induce the large-scale degree that generally suppresses with the chemotactic activity that reaches the influence condition culture medium of the M Φ chemoattractant in M Φ-source.

The test of further second chemotaxis demonstrate be suppressed towards macrophage chemotaxis from the supernatant of the macrophage of Chemerin140-mice, Chemerin15-mice and Chemerin17-mice-processing.These results show that the macrophage with C 15m and C17m pre-treatment and activation makes the amount and/or the biological activity of the chemoattractant that discharged by macrophage reduce, so these peptides can reduce significantly to the monocyte/macrophage that continues of inflammation part and raise.

The conditioned medium of the macrophage that uses use by oneself C140+LPS/IFN γ, C15m+LPS/IFN γ, C17m+LPS/IFN γ and only handle with LPS/IFN γ in second chemotaxis test is with the inhibition relevant potential pathologic-physiological reaction (repercussions) of assessment with the macrophage activation of passing through C140 and C-terminal peptide thereof.

Make cell (0.5 * 10 ⁶) chemoattractant (Kai Morui peptide) in the base apertures of Boyden chamber of improvement or conditioned medium migration be above 4 hours.Serum-free medium (SFM) is as negative control.Filter is fixed in 4% formaldehyde, subsequently with DAPI with nucleus dyeing and make it visual.Block diagram shows average Migration Index (chemoattractant % threshold zone/SFM% the threshold zone) ± SEM for each processed group.Each at least three hole of post representative, 6 width of cloth figure are got in each processing.Except as otherwise noted, p＜0.001***; P＜0.01** significance is with respect to LPS/IFN γ conditioned medium.AB is meant anti--Mus Kai Morui antibody.

The result who from Fig. 5, represents as can be seen, macrophage-conditioned medium itself does not show the chemotactic activity for macrophage, however the macrophage culture medium of LPS/IFN γ-processing induces that macrophage is chemotactic to significantly improve.In addition, macrophage shows towards the chemotaxis from the conditioned medium of the macrophage of C 140+LPS/IFN γ, C 15m+LPS/IFN γ and C 17m+LPS/IFN γ-processing and reduces, and shows that C140, C15m and C17m induce the ability that generally suppresses of macrophage chemoattractant on a large scale.

For the supernatant of getting rid of Kai Morui-processing comprises the probability of Kai Morui-deutero-chemotactic protein/peptide, before the huge cell migration of biting of assessment with supernatant with Zhong He Kai Morui antibody hatch.Kai Morui does not have to show the migration that helps in the supernatant of Kai Morui-processing.

The Chemerin15-mice suppresses zymosan-inductive peritonitis

Peritonitis can be by causing acute inflammatory response zymosan granule (yeast cell wall composition) peritoneal injection and induced.It is that monocytic known time dependence assembles that (summary is referring to Lawrence T et al.Nat Rev Immunol.2002 October then that zymosan-inductive peritonitis is followed in the mouse peritoneum chamber neutrophilic leukocyte; 2 (10): 787-795).This model has been used to prove definite medium (mediator) lipoxin A 4 (Lipoxin A4) and annexin-1 is preceding-dissolving (pro-resolving) characteristic, and this characteristic is accompanied by organizational structure and the recovery of function and neutrophilic leukocyte and monocytic inhibition of oozing out and shorten the time-histories of inflammation early usually.Zymosan A granule (10 μ g-1mg) (Taylor PR et al.Eur J Immunol.2005 July of range of doses has been used in the previous experiment of reporting in the document; 35 (7): 2163-2174; AritaM et al.J.Biol.Chem.2006 August 11; 281 (32): 22847-22854).

Consider the high chemotactic potential of Kai Morui and for proteoclastic intrinsic demand, use the terminal synthetic peptide chemerin15 of C-that the antiinflammatory action in the aseptic peritonitis model is carried out characterizing in the body, this is because C15 lacks chemotactic activity (Fig. 3 and Fig. 4 B) to a great extent, but also plays the antiinflammatory action (table 1) that is equivalent to proteoclastic Kai Morui.

With reference to the result shown in Fig. 6,10 μ g/ mices (each resident macrophage 1-2 granule) are used in this research, because this is considered to closer represent Pathophysiology dosage.

More specifically, male C57B 16/J mice (8-12 week) was then injected 0.5ml PBS or zymosan (2 * 10 by peritoneal injection 0.5mlPBS or 0.5ml Chemerin15-mice (0.32ng/kg) after 1 hour ⁶Individual granule/chamber).After 4 hours, with sacrifice of animal, with 5ml PBS-3mM EDTA flushing peritoneal cavity.Utilize trypan blue to get rid of test and obtain total cell count.Form (neutrophilic leukocyte and mononuclear phagocyte) in order to measure cell, with 2.4G2 anti--FcgRII/III mAB pair cell blocking-up (block) 5 minutes and with PE-conjugated anti--mice Ly-6G and FITC-conjugated anti--mice 7/4mAB dyeing 10 minutes.Before carrying out facs analysis with CellQuest software with cell fixation in 1% formaldehyde.Make up door (gate) around two groups, these two groups are neutrophilic leukocyte (N; 7/4 ^High, Ly-6G ^High) and inflammatory mononuclear cell (Mo; 7/4 ^High).C15 is meant the Chemerin15-mice.With respect to zymosan-processing, Z is meant zymosan, p＜0.01**; P＜0.05*.

Neutrophilic leukocyte (7/4 ^High, Ly-6G ^High), mononuclear cell (7/4 ^High, Ly-6G ^Low) and resident macrophage group (7/4 ^Low, Ly-6G ^Low) according to Gordon S and Taylor PR NatRev Immunol.2005; 5 (12): 953; Taylor PR et al.Eur J Immunol.2003 August; 33 (8): 2090-2097; With Taylor PR et al.Eur J Immunol.2005 July; 35 (7): 2163-2174 determines.

This result of study shows that mononuclear cell and neutrophil recruitment that the mice of handling with the C15m of 0.32ng/kg (8pg/ mice) dosage shows zymosan-initiation reduce by 42% and 52% (Fig. 6) respectively.The level of TNF α also reduces in the mice of handling with C15m.

The above results is further studied.In order to determine C15 peptide anti-inflammatory property in vivo, continue to surpass 48 hours and carry out the time-histories experiment.By facs analysis according to disclosed testing program (Taylor, P.R.et al., (2005) Eur J Immunol 35:2163-2174; Taylor, P.R., (2003) .Eur J Immunol 33:2090-2097) determine the neutrophilic leukocyte (7/4 in the peritoneal lavage ^High, Ly-6G ^High) and mononuclear cell (7/4 ^High, Ly-6G ^Low) group.Zymosan is administered to the mouse peritoneum intracavity and produces inflammatory cell and extravasate into time-dependent in the peritoneal cavity, and it follows the typical characteristic (Fig. 7 A-schemes B, solid line) that acute inflammation is replied.Neutrophilic leukocyte was first leukocyte that infiltrates into intracavity, can be after giving zymosan detects in 2 hours, and leukocyte reached peak value (1.95 * 10 at 4 hours ⁶Individual cell).The peritoneal cavity that mononuclear cell flows into inflammation can detect (0.69 * 10 first after 4 hours ⁶Individual cell), behind the injection zymosan, reached peak value (1.25 * 10 in 24 hours ⁶Individual cell), descend thereafter.Excite the C15 pretreatment with 8pg/ mice (≈ 0.32ng/kg) dosage in preceding 1 hour at zymosan, make neutrophilic leukocyte advance to 2 hours and reach peak value, be about the mice that zymosan excites the peak value size 50% (from 1.25 * 10 ⁶Drop to 0.62 * 10 ⁶Individual cell; Fig. 7 A, dotted line).Can observe the remarkable inhibition of C15 at 2 hours (50%), 4 hours (66%) and 4 hours (50%) to neutrophilic infiltration.The peritoneum monocytic quantitative aspects of the C15 peptide of the single dose of 8pg in the chamber of the inflammation that reduces all time points also was effectively, 4 hours (63%), 8 hours (61%) and 48 hours (64%; Fig. 7 B, dotted line) can be observed and surpass 60% inhibition.Monocyte infiltration ratio 2-4 hour in zymosan injection back is the highest by (0.51 * 10 ⁶/ h), the ratio (0.18 * 10 in the chamber that has reduced the inflow inflammation of C 15 ⁶/ h).Therefore, the C15 peptide of the single dose before the zymosan-excite provides the remarkable protection at zymosan-inductive peritoneum inflammation, surpasses 48 hours persistent period of experiment.

The proper time point that the anti-inflammatory activity that 4 hours time points are C15 behind the time-histories experimental identification zymosan comes into force.In this research, the decline of the neutrophilic leukocyte of the C15 generation zymosan-initiation of single dose and the dose dependent of monocyte recruitement, this raises (the ≈ 0.32ng/kg at 8pg/ mice C15; Fig. 7 C-Fig. 7 E and Figure 16 A-Figure 16 B) time maximum.When C15 when zymosan-excite preceding 1 hour is given, neutrophilic leukocyte quantity is from 1.9 * 10 ⁶Reduce to 0.78 * 10 ⁶(reduce 63%; Fig. 7 C), the mononuclear cell level is from 0.69 * 10 ⁶Reduce to 0.30 * 10 ⁶(reduce 62%; Fig. 7 D has illustrated representational FACS figure at 4 hours time points among Fig. 7 E).Give C15 and also reduced the expression of preceding-inflammatory cytokine in the peritoneal lavage when 4h significantly, comprise TNF α (51%), L-1 β (67%), IL-6 (67%), MCP-1 (59%) and KC (38%; Fig. 7 F).Also find with the same dose of C15 and the time when giving in the mesosome, lacking active control peptide C15-S of extracorporeal anti-inflammatory and C15-M (table 1) is not protectiveness, this judges (Fig. 7 C-Fig. 7 D) by mononuclear cell and neutrophilic leukocyte level.When injecting the back C15 that gave same dose in 2 hours at zymosan, give behind the zymosan still to can be observed in 4 hours mononuclear cell (0.69 * 10 ⁶To 0.42 * 10 ⁶Individual cell; Reduce 42%) and neutrophil recruitment (1.9 * 10 ⁶To 0.83 * 10 ⁶Individual cell; Minimizing 60%) remarkable inhibition (Fig. 7 G).This shows that C15 can reduce neutrophilic leukocyte and monocyte recruitement down in the inflammatory situation of having set up (setting), and provides the C15/C15-derivant can represent another indication of the noticeable pharmacophore of targeting inflammatory condition of illness.

The blocking-up of endogenous Kai Morui material worsens the peritoneum inflammation

By excite at zymosan preceding 4 hours or 24 hours in the mice i.p injection and monoclonal anti-rmChemerin antibody (ChAb) or contrast IgG 1h study potential inherent the acting on of Kai Morui and Kai Morui-deutero-peptide.The previous ChAb that finds can vitro inhibition C15 and Kai Morui-inductive M Φ chemotaxis and antiinflammatory action, and contrast IgG does not possess this effect (Fig. 8 A-Fig. 8 B).In vivo, the mice that discovery is handled with respect to contrast IgG-, the neutralization of endogenous Kai Morui material causes increasing by 63% and the mononuclear cell level improves 45% in the quantity of 4 hours time point peritoneums neutrophilic leukocyte, and injects peritoneum neutrophilic leukocyte in back 24 hours and monocytic level improves 170% and 86% (Fig. 8 C-Fig. 8 D) at zymosan.The peritoneum inflammation increases the weight of the endogenous antiinflammatory action that shows that the Kai Morui material is important in vivo in 24 hours.

But only use the Chemerin15-mice not induce neutrophilic leukocyte or macrophage to raise and reduce the TNF alpha levels

Male C57B 16/J mice (8-12 week) is through peritoneal injection 0.5ml PBS or 0.5ml Chemerin15-mice | (0.32ng/kg).After 4 hours, with three sacrifice of animal of each processed group, with 5ml PBS-3mM EDTA flushing peritoneal cavity.Utilize trypan blue to get rid of test and obtain total cell count.In order to determine that cell forms (neutrophilic leukocyte and mononuclear phagocyte), with 2.4G2 anti--FcgRII/III mAB with cell blocking-up (block) 5 minutes and with PE-conjugated anti--mice Ly-6G and FITC-conjugated anti--mice 7/4mAB dyeing 10 minutes.Before carrying out facs analysis with CellQuest software with cell fixation in 1% formaldehyde.Make up door (gate) around two groups, these two groups are neutrophilic leukocyte (N; 7/4 ^High, Ly-6G ^High) and inflammatory mononuclear cell (Mo; 7/4 ^High, Ly-6G ^Low).C15 is meant the Chemerin15-mice.Handle p＜0.01** with respect to PBS-.Ns is meant significant difference p＞0.05 that does not have on the statistics.

Result from Fig. 9 as can be seen, the C15m of 0.32ng/kg can not cause mononuclear cell or neutrophilic leukocyte the migration.Yet, observe the remarkable minimizing of TNF α.

This model of research mice aseptic peritonitis is widely used in experimental medicine and pharmacology, the light inflammation that representative is caused by moderate tissue injury or infection.Described result shows that C15m can cross the effect of realization therapeutic antiinflammatory.

The expression of Rantes and TNF alpha transcriptional thing in the human inhibition of the Chemerin13-that the modifies mouse macrophage

Cultivate Mus peritoneal macrophages (PM θ) under various conditions: untreated, LPS (100ng/ml) and IFN γ (20ng/ml), 15 hours, the Chemerin13-mankind of modification (1nM) pretreatment 1 hour, LPS/IFN γ is 15 hours subsequently.Block diagram show determine by qRT-PCR and be standardized as house-keeping gene (house-keeping gene, housekeeper), the average expression of the cytokine transcript of hypoxanthine phosphoribosyltransferase (HPRT).For each processing, experiment is carried out with in triplicate mensuration, and n=1 independently tests.For the sample of LPS/IFN γ-t processing, unless otherwise noted, p＜0.01**; P＜0.05*.The sequence of the C13h peptide of modifying is NH ₂-FHSFYFPGQFAFS-COOH (Seq ID No:43)-in this sequence, the N-terminal P among the C13h is replaced by aminoacid F, and therefore this peptide is called as the C13h of modification.

Result from Figure 10 as can be seen, the C13h of modification significantly reduces the expression of TNF α and RANTES.

Chemerin 17-mice does not influence the inductive macrophage chemotaxis of C140

Stimulating peritoneum after 4 days, raise PM θ with the BioGEL pearl.Peritoneal cavity with the male C57B16/J mice of 5ml PBS-2mMEDTA lavation.Cell is through centrifugal and be resuspended among the RPMI that is supplemented with 0.5%BSA and 25mM Hepes.Make cell (0.5 * 10 ⁶) chemoattractant (C140, C17m or C17m+C140) migration in base apertures is above 4 hours.Filter is fixed in 4% formaldehyde, dyes with DAPI pair cell nuclear subsequently and make it visual.Serum-free medium is as negative control (/-).Cell before chemotaxis test with pertussis toxin, PT (PTX) preincubate 30 minutes.Block diagram among Figure 11 shows the average Migration Index ± SEM of each processed group.Each at least three hole of post representative and each are handled and are got 3 width of cloth figure at least.Unless otherwise noted, for the hole that SFM handles, p＜0.001***; P＜0.01**; P＜0.05*.

Result among Figure 11 shows that C17m and C140's gives jointly not show that influencing macrophage moves to C140.

Chemerin15-mice and Chemerin17-mice suppress by stimulate the TNF α secretion of mouse macrophage with zymosan

Under multiple condition, cultivate PM θ: untreated; Zymosan 15h; 1 hour+zymosan of Kai Morui (1pM) pretreatment 15 hours.Block diagram shows the average expression of the TNF α that measures by ELISA ± SEM.For each processing, experiment is carried out with in triplicate mensuration.The representative data that adopts from three independent experiments of different donorcellses has been shown among Figure 12.For the sample of zymosan-processing, p＜0.001***; P＜0.01**.Dexa is meant dexamethasone (1mM), and nd is meant to be lower than and detects lower limit (0.25ng/ml).

As can be seen, can suppress zymosan-inductive TNF with C15m (1pM) and C17m (1pM) processing and express (C15m; 21%, C17m; 30%).Therefore, C15m and C17m suppress by antibacterial (LPS) and the inductive macrophage activation of yeast (zymosan A).

Chessboard (Checkerboard) the analysis showed that Chemerin140 and Chemerin15-mice induce the macrophage chemotaxis but not chemokinesis

The chessboard analysis allows to distinguish chemotaxis and chemokinesis.Chemotaxis is by shown in the migration of the chemoattractant of lower Kong Zhongxiang higher concentration.Chemokinesis is meant that non-directional cell movement increases, and it takes place with the Concentraton gradient that exists irrelevant.The chessboard analysis is performed such: with cell with C140 (10-500pM) or C15m (10-1000pM) preincubate, and make they in lower hole respectively towards C140 (10-1000pM) or C15m (10-1000pM) migration to form the chessboard of concentration.

More specifically, stimulating peritoneum to raise PM θ after 4 days with the BioGEL pearl.Peritoneal cavity with the male C57B16/J mice of 5mlPBS-2mM EDTA lavation.Cell is through centrifugal and be resuspended among the RPMI that is supplemented with C57B16/J.Before chemotaxis test with cell (0.5 * 10 ⁶) hatched 30 minutes with C140 or C15m, it is moved above 4 hours towards chemoattractant in base apertures.Filter is fixed in 4% formaldehyde, dyes with DAPI pair cell nuclear subsequently and make it visual.Serum-free medium (SFM) is as negative control (/-), and CC chemotactic factor RANTES is as positive control (25ng/ml).Block diagram among Figure 13 shows the average Migration Index ± SEM for each processed group.Each at least three hole of post representative and each are handled and are got 3 width of cloth figure at least.With respect to the hole that SFM-handles, p＜0.001***.

Have been found that C140 and C15m cause real chemotaxis rather than chemical activity when migrating into the bottom hole of Boyden chamber, this migration takes place when only having the chemoattractant of higher concentration therein, does not then take place when the upside of filter has the chemoattractant of higher concentration.

C15m be shown as than a little less than the C140 the chemotactic decoy of macrophage of Duoing.

C15 induces the macrophage phagocytic effect of zymosan

For by macrophage at external identification zymosan, by with the ice-cold 2mM EDTA lavation among the PBS from handle with Biogel pearl (2%w/v) preceding 4 days the intraperitoneal mice of handling isolating the peritoneal effusion cell.With 2.5 * 10 ⁵The density of individual cells/well is tiled in macrophage in 24 orifice plates in the Optimen culture medium.In the presence of 0.1pM, 1pM, 10pM, 100pM or 1nM Chemerin15, in identification test, cell washes cell three times with culture medium before at the zymosan (Invitrogen) that adding is added with Fluorescein isothiocyanate (FITC)-labelling, and macrophage/granule ratio is 10: 1.Carrier=the do not have control sample of Chemerin15.Carry out facs analysis after the picked-up of FITC-zymosan, the picked-up of FITC-zymosan is expressed as relative discrimination index, that is the ratio of the geometric mean of the macrophage of the geometric mean/usefulness vehicle treated of the macrophage of the ratio of the % cell of picked-up zymosan * C15 processing.

Result shown in Figure 15 shows that Chemerin15 induces the macrophage phagocytic of zymosan.The macrophage phagocytic effect induce maximum when Chemerin15 concentration is 10pM.These results show that the Kai Morui peptide can be by increasing macrophage engulfing and accelerate wound repair apoptotic cell (apoptotic cell), cell debris, pathogen and pathogen product.

Discuss

The collaborative acutely inflamed primary event of multiple medium is known.For example, the eicosanoid in lipid-source (eicosanoids), cytokine and chemotactic factor are regulated the blood vessel variation and inflammatory cell is raised.Before-inflammatory cytokine, comprise the signal pipeline in TNF α and the IL-1 γ activation endotheliocyte, cause the rise of adhesion molecule expression, promote catching of circulating leukocyte.The result who above points out shows all the components that can suppress inflammatory response derived from the C-terminal peptide of Chemerin140.The result shows that also the C-terminal peptide derived from Chemerin140 can reduce chemotactic factor level and can be with the therapeutic agent that acts on endotoxin shock.

All peptides that use in this research are Kai Morui-deutero-, and demonstrate fabulous high-effect (10 ^-12M), thisly high-effectly guarantee that these media join the chemotaxin in complement-source, C5a des-arg (10 ^-12M), formyl-methionyl-leucyl-phenylalanine (fMLP; 10 ^-11M), leukotriene B4 (LTB4; 10 ^-11M), TNF α (10 ^-11M), LPS (10 ^-15M) and IL-1 (10 ^-14M) in the ranks.The applicant knows does not also have pharmaceutical preparation to be proved to be 10 ^-11M-10 ^-15M shows pharmacological action.In fact, dexamethasone gives so that the concentration of micro-molar range is external usually, and locates to realize 50% downward modulation of mononuclear cell and neutrophilic leukocyte inflow in zymosan-inductive peritonitis model 30 μ g/ mices (1.2mg/kg).The Chemerin15-mice is lowered to raising of mononuclear cell and neutrophilic leukocyte and the similar degree of 30 μ g dexamethasone.In this Mus inflammatory model, the Chemerin15-mice just can produce suitable antiinflammatory action with the dosage of 8pg/ mice (0.32ng/kg) only.

Second chemotaxis test makes chemotactic potential from the supernatant of macrophage activation test by quantification, and has determined the influence of the chemotactic factor inhibition of Kai Morui-mediation to medium chemotactic characteristic.These results' analysis has disclosed with independent LPS/IFN γ and has compared, and macrophage descends to the migration from the supernatant of the macrophage of Kai Morui+LPS/IFN γ-processing, and this has shown generally suppressing of macrophage chemoattractant on a large scale.The embodiment that provides shows, compares with C140, and the chemoattractant characteristic of the anti-inflammatory peptides in Kai Morui-source is limited or does not possess the chemoattractant characteristic.

In a word, the result shows that the C-terminal peptide of Kai Morui all shows very strong antiinflammatory performance in vitro and in vivo.

Material and method

Animal

All zooscopies have all obtained local ethics approval and have carried out according to British Home Office's rule (animal law, scientific procedure bill (Guidance on the Operation of Animals, Scientific Procedures Act), 1986).

Antibody and reagent

Anti-people Kai Morui, anti-Mus Chemerin AB, hChemerin 137 (serial ID no:31 can be used as recombinant Glu21-Ser157 and obtains from RandD), mChemerin 140 (SeqID no:34), anti--mRANTES Capture AB, anti--mRANTES Detection AB, mRANTES, mTNF α, anti--mTNF α Capture AB, anti--mTNF α DetectionAB are all available from R﹠amp; D Systems.Kai Morui peptide (ClIm, C13m, C13h, C15m, C17m) is synthesized (www.biosyn.com) by biosynthesis.Dexamethasone, lipopolysaccharide (escherichia coli (E.coli)), leupeptin obtain from Sigma Aldrich.Interferon gamma (IFN γ) is available from Peprotech.The OPD sheet obtains from Dakocytomata, and streptavidin (Streptavidin)-HRP and the agent of StrepAv-HRP dilution buffer are available from Endogen.Luminex 6-plex test kit (IL-12p40, IL-1 β, IL-6, MCP-1, TNF α IL-10) is provided by Bio-rad, and utilizes Bio-rad analyser and X software analysis.

The inhibition of macrophage activation-macrophage activation test

1ml 2%BioGEL polyacrylamide pearl peritoneal injection (ip) in sterile phosphate-buffer saline (PBS) is gone into the C57B1/6J mice.Ip BioGEL gives back four days, passes through CO according to British Home Office's criterion ₂Method is put to death mice.Wash peritoneal cavity to obtain the Premeabilisation of cells thing that BioGEL-causes/causes with the aseptic PBS-2mM EDTA of 10ml.The suspension of the cell that obtains 4 ℃ at 1000xg centrifugal 5 minutes.Abandon supernatant, cell precipitation is resuspended in the 6mls OptiMEM culture medium that is supplemented with 2mM glutamine, 50 units/ml penicillin and 50 μ g/ml streptomycins.(hatch and utilize the haemocytometer quantification after 5-10 minute with spy gram by the solution of Turk ' s) on ice for macrophage.With cell suspending liquid (2mls; 1.5 * 10 ⁶/ hole) be tiled in six hole tissue culturing plates (diameter is 35mm:Costar, UK) in, and make it comprise 5%CO at 37 ℃ ₂Moistening atmosphere in adhere to 2 hours to separate the macrophage group by adhering to.By cell centrifugation smear (cytospinning), dye and based on morphocytology counting, this has obtained the purity greater than 95% with methylene blue and eosin pair cell.Not adherent cell (being mainly granulocyte) is dropped, and the hole is washed three times to remove loose cell that adheres to or dead cell with aseptic PBS.For the expression decreased of the potential inhibition of assessing macrophage activation and preceding-inflammatory mediator of bringing thus, with macrophage (1.5 * 10 ⁶Cells/well) with Kai Morui peptide (C11m, C13m, C15m, C17m; 10 ^-12-10 ^-8M) or positive control (dexamethasone; 1 μ M) preincubate 1 hour together uses LPS (100ng/ml) and IFN γ (20ng/ml) to excite 15 hours subsequently.In order to determine it to the sensitivity of PTX with to proteoclastic dependency, with cell with PTX (200ng/ml) or leupeptin (15 μ g/ml) preincubate.Other cell is only handled with peptide.Collect supernatant, be stored in-20 ℃ to be used for elisa (ELISA) and Luminex experiment.With lysis to extract total RNA by the TRIZOL method.Lysate is stored in-80 ℃, extracts RNA according to the Guide Book (Qiagen, RNeasy Mini Prep Kit) of manufacturer.

Detect excretory albumen by ELISAs and Luminex

Concentration by RANTES, tumor necrosis factor (TNF α) and CCL9 in the ELISA assessment cell conditioned medium liquid.(Bio-rad micropearl array more than 6 method (Bio-rad 6 plexassay)) determine IL-12p40, IL-10, IL-1 β, TNF α, MCP-1 (mononuclear cell chemoattractant albumen-1) and IL-6 level by Luminex streaming microballon method (Luminexmultiplex bead assay).Be limited to 0.1-0.5ng/ml under the detection of ELISA, be limited to 10-50pg/ml under the detection of Luminex method.

RNA preparation and RT-PCR

Utilize Qiagen RNeasy test kit to extract total RNA, utilize the Sybr-Green method to make it be inverted record and stand qRT-PCR.Utilize 2-AACT methods analyst data (Livak, KJ.﹠amp; Schmittgen T.D. (2001), Methods 25:402-408).

The chemotaxis test

Assess cell migration by utilizing open-work film (ChemoTX, 6-mm diameter, 8-μ m aperture).In brief, collect the cell that BioGEL-causes and being placed on the open-work film among the RPMI that is supplemented with 25mMHepes and 0.1% bovine serum albumin (250000 cell/films).Make cell towards Kai Morui peptide (1pM-100nM) migration 4 hours.By before cell being placed on the open-work film 30 minutes with cell and pertussis toxin, PT (PTX, 200ng/ml, Sigma-Aldrich) signal transduction blocked via g protein coupled receptor in 30 minutes of preincubate together.Cell in the migration of the downside of film is fixed in (3% formaldehyde) and dyes with DAPI.Migration is the pixel count in the painted nucleus of DAPI under the confocal microscope (2 photos/film, each handles at least 3 parallel holes) quantitatively.The percentage threshold zone (TA) that utilizes MetamorphOffline software analysis image to occupy with the cell of determining to be moved.Handle TA serum-free medium TA acquisition migration indicant (migration indices) by dividing.For the test of second chemotaxis, use ChemoTx 3-mm diameter, 8-μ m pore membrane with 50000 cells/film.

Mus peritonitis

The C57BL6/J mice gives preceding 1 hour i.p. of 500 μ l, 10 μ g zymosan A at i.p. and gives 500 μ l Chemerin15-mices (0.32ng/kg) or only give carrier (aseptic PBS).With after its humanity execution, collect peritoneal effusion after 4 hours by carrying out peritoneal lavage with the aseptic PBS-3mM EDTA of 5ml.Obtain acellular irrigating solution to be used for ELISA, preparation exudate cell is to be used for following analysis.

Difference numeration of leukocyte and facs analysis

The C57BL6/J mice gives preceding 1 hour i.p. of 500 μ l, 10 μ g zymosan A at i.p. and gives 500 μ l Chemerin15 (0.32ng/kg) or carrier (PBS).After 2 hours, 4 hours, 8 hours, 16 hours, 24 hours and 48 hours, its humanity is put to death.The aliquot of preparation bal cell is measured to be used for numeration of leukocyte total and difference.In order to determine cell composition (PMN and mononuclear cell), with anti--mice 2.42G Fc μ II/III (0.5 μ g/0.1 * 10 ⁶Individual cell) blocking-up cell 10 minutes, and with FITC-conjugated anti--mice 7/4 and PE-is conjugated resists-mice Ly-6G (0.5 μ g/0.5 * 10 ⁶Individual cell; Clone rmC5-3 and RB6-8C5 are respectively from BD Pharmingen) dyeing (10 minutes).On the FACSCalibur flow cytometer, analyze with CellQuest software pair cell.For each sample, 10,000 incidents of minimum acquisition.Make up door around three groups, these three groups are neutrophilic leukocyte (7/4 ^High, Ly-6G ^High), mononuclear cell (7/4 ^High, Ly-6G ^Low) and resident macrophage (7/4 ^Low, Ly-6G ^Low).Measure the total incident percentage ratio in each group.In addition, collect acellular irrigating solution to be used for ELISA and Luminex test.

Statistics

Utilize GraphPad Prism software to carry out Xue Shengshi t check and monobasic ANOVA.

The P28328BKB-sequence table

Sequence table

＜110〉ISIS Innovaton Ltd. (ISIS Innovation Limited)

＜120〉treatment of inflammation and/or endotoxin shock

<130>P28328BKB

<140>PCT/GB2008/001020

<141>2008-0325

<150>GB?0705488.5

<151>2007-03-22

<160>45

<170>PatentIn?version?3.3

<210>1

<211>11

<212>PRT

<213>Mus?sp.

<400>1

Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala

1 5 10

<210>2

<211>12

<212>PRT

<213>Mus?sp.

<400>2

Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10

<210>3

<211>13

<212>PRT

<213>Mus?sp.

<400>3

Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe?Ser

1 5 10

<210>4

<211>15

<212>PRT

<213>Mus?sp.

<400>4

Ala?Gly?Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala

1 5 10 15

<210>5

<211>16

<212>PRT

<213>Mus?sp.

<400>5

Ala?Gly?Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10 15

<210>6

<211>17

<212>PRT

<213>Mus?sp.

<400>6

Ala?Gly?Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10 15

Ser

<210>7

<211>12

<212>PRT

<213>Mus?sp.

<400>7

Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala

1 5 10

<210>8

<211>13

<212>PRT

<213>Mus?sp.

<400>8

Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala

1 5 10

<210>9

<211>14

<212>PRT

<213>Mus?sp.

<400>9

Gly?Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala

1 5 10

<210>10

<211>13

<212>PRT

<213>Mus?sp.

<400>10

Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10

<210>11

<211>14

<212>PRT

<213>Mus?sp.

<400>11

Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10

<210>12

<211>15

<212>PRT

<213>Mus?sp.

<400>12

Gly?Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10 15

<210>13

<211>14

<212>PRT

<213>Mus?sp.

<400>13

Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe?Ser

1 5 10

<210>14

<211>15

<212>PRT

<213>Mus?sp.

<400>14

Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe?Ser

1 5 10 15

<210>15

<211>16

<212>PRT

<213>Mus?sp.

<400>15

Gly?Glu?Asp?Pro?Gis?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe?Ser

1 5 10 15

<210>16

<211>11

<212>PRT

<213>Homo?sapiens

<400>16

Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala

1 5 10

<210>17

<211>12

<212>PRT

<213>Homo?sapiens

<400>17

Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10

<210>18

<211>13

<212>PRT

<213>Homo?sapiens

<400>18

Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe?Ser

1 5 10

<210>19

<211>15

<212>PRT

<213>Homo?sapiens

<400>19

Ala?Gly?Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala

1 5 10 15

<210>20

<211>16

<212>PRT

<213>Homo?sapiens

<400>20

Ala?Gly?Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10 15

<210>21

<211>17

<212>PRT

<213>Homo?sapiens

<400>21

Ala?Gly?Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10 15

Ser

<210>22

<211>12

<212>PRT

<213>Homo?sapiens

<400>22

Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala

1 5 10

<210>23

<211>13

<212>PRT

<213>Homo?sapiens

<400>23

Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala

1 5 10

<210>24

<211>14

<212>PRT

<213>Homo?sapiens

<400>24

Gly?Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala

1 5 10

<210>25

<211>13

<212>PRT

<213>Homo?sapiens

<400>25

Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10

<210>26

<211>14

<212>PRT

<213>Homo?sapiens

<400>26

Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10

<210>27

<211>15

<212>PRT

<213>Homo?sapiens

<400>27

Gly?Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe

1 5 10 15

<210>28

<211>14

<212>PRT

<213>Homo?sapiens

<400>28

Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe?Ser

1 5 10

<210>29

<211>15

<212>PRT

<213>Homo?sapiens

<400>29

Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe?Ser

1 5 10 15

<210>30

<211>16

<212>PRT

<213>Homo?sapiens

<400>30

Gly?Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe?Ser

1 5 10 15

<210>31

<211>137

<212>PRT

<213>Homo?sapiens

<400>31

Glu?Leu?Thr?Glu?Ala?Gln?Arg?Arg?Gly?Leu?Gln?Val?Ala?Leu?Glu?Glu

1 5 10 15

Phe?His?Lys?His?Pro?Pro?Val?Gln?Trp?Ala?Phe?Gln?Glu?Thr?Ser?Val

20 25 30

Glu?Ser?Ala?Val?Asp?Thr?Pro?Phe?Pro?Ala?Gly?Ile?Phe?Val?Arg?Leu

35 40 45

Glu?Phe?Lys?Leu?Gln?Gln?Thr?Ser?Cys?Arg?Lys?Arg?Asp?Trp?Lys?Lys

50 55 60

Pro?Glu?Cys?Lys?Val?Arg?Pro?Asn?Gly?Arg?Lys?Arg?Lys?Cys?Leu?Ala

65 70 75 80

Cys?Ile?Lys?Leu?Gly?Ser?Glu?Asp?Lys?Val?Leu?Gly?Arg?Leu?Val?His

85 90 95

Cys?Pro?Ile?Glu?Thr?Gln?Val?Leu?Arg?Glu?Ala?Glu?Glu?His?Gln?Glu

100 105 110

Thr?Gln?Cys?Leu?Arg?Val?Gln?Arg?Ala?Gly?Glu?Asp?Pro?His?Ser?Phe

115 120 125

Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe?Ser

130 135

<210>32

<211>143

<212>PRT

<213>Homo?sapiens

<400>32

Glu?Leu?Thr?Glu?Ala?Gln?Arg?Arg?Gly?Leu?Gln?Val?Ala?Leu?Glu?Glu

1 5 10 15

Phe?His?Lys?His?Pro?Pro?Val?Gln?Trp?Ala?Phe?Gln?Glu?Thr?Ser?Val

20 25 30

Glu?Ser?Ala?Val?Asp?Thr?Pro?Phe?Pro?Ala?Gly?Ile?Phe?Val?Arg?Leu

35 40 45

Glu?Phe?Lys?Leu?Gln?Gln?Thr?Ser?Cys?Arg?Lys?Arg?Asp?Trp?Lys?Lys

50 55 60

Pro?Glu?Cys?Lys?Val?Arg?Pro?Asn?Gly?Arg?Lys?Arg?Lys?Cys?Leu?Ala

65 70 75 80

Cys?Ile?Lys?Leu?Gly?Ser?Glu?Asp?Lys?Val?Leu?Gly?Arg?Leu?Val?His

85 90 95

Cys?Pro?Ile?Glu?Thr?Gln?Val?Leu?Arg?Glu?Ala?Glu?Glu?His?Gln?Glu

100 105 110

Thr?Gln?Cys?Leu?Arg?Val?Gln?Arg?Ala?Gly?Glu?Asp?Pro?His?Ser?Phe

115 120 125

Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe?Ser?Lys?Ala?Leu?Pro?Arg?Ser

130 135 140

<210>33

<211>163

<212>PRT

<213>Homo?sapiens

<400>33

Met?Arg?Arg?Leu?Leu?Ile?Pro?Leu?Ala?Leu?Trp?Leu?Gly?Ala?Val?Gly

1 5 10 15

Val?Gly?Val?Ala?Glu?Leu?Thr?Glu?Ala?Gln?Arg?Arg?Gly?Leu?Gln?Val

20 25 30

Ala?Leu?Glu?Glu?Phe?His?Lys?His?Pro?Pro?Val?Gln?Trp?Ala?Phe?Gln

35 40 45

Glu?Thr?Ser?Val?Glu?Ser?Ala?Val?Asp?Thr?Pro?Phe?Pro?Ala?Gly?Ile

50 55 60

Phe?Val?Arg?Leu?Glu?Phe?Lys?Leu?Gln?Gln?Thr?Ser?Cys?Arg?Lys?Arg

65 70 75 80

Asp?Trp?Lys?Lys?Pro?Glu?Cys?Lys?Val?Arg?Pro?Asn?Gly?Arg?Lys?Arg

85 90 95

Lys?Cys?Leu?Ala?Cys?Ile?Lys?Leu?Gly?Ser?Glu?Asp?Lys?Val?Leu?Gly

100 105 110

Arg?Leu?Val?His?Cys?Pro?Ile?Glu?Thr?Gln?Val?Leu?Arg?Glu?Ala?Glu

115 120 125

Glu?His?Gln?Glu?Thr?Gln?Cys?Leu?Arg?Val?Gln?Arg?Ala?Gly?Glu?Asp

130 135 140

Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe?Ser?Lys?Ala?Leu

145 150 155 160

Pro?Arg?Ser

<210>34

<211>140

<212>PRT

<213>Mus?sp.

<400>34

Thr?Val?Gly?Thr?Glu?Pro?Glu?Leu?Ser?Glu?Thr?Gln?Arg?Arg?Ser?Leu

1 5 10 15

Gln?Val?Ala?Leu?Glu?Glu?Phe?His?Lys?His?Pro?Pro?Val?Gln?Leu?Ala

20 25 30

Phe?Gln?Glu?Ile?Gly?Val?Asp?Arg?Ala?Glu?Glu?Val?Leu?Phe?Ser?Ala

35 40 45

Gly?Thr?Phe?Val?Arg?Leu?Glu?Phe?Lys?Leu?Gln?Gln?Thr?Asn?Cys?Pro

50 55 60

Lys?Lys?Asp?Trp?Lys?Lys?Pro?Glu?Cys?Thr?Ile?Lys?Pro?Asn?Gly?Arg

65 70 75 80

Arg?Arg?Lys?Cys?Leu?Ala?Cys?Ile?Lys?Met?Asp?Pro?Lys?Gly?Lys?Ile

85 90 95

Leu?Gly?Arg?Ile?Val?His?Cys?Pro?Ile?Leu?Lys?Gln?Gly?Pro?Gln?Asp

100 105 110

Pro?Gln?Glu?Leu?Gln?Cys?Ile?Lys?Ile?Ala?Gln?Ala?Gly?Glu?Asp?Pro

115 120 125

His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe?Ser

130 135 140

<210>35

<211>146

<212>PRT

<213>Mus?sp.

<400>35

Thr?Val?Gly?Thr?Glu?Pro?Glu?Leu?Ser?Glu?Thr?Gln?Arg?Arg?Ser?Leu

1 5 10 15

Gln?Val?Ala?Leu?Glu?Glu?Phe?His?Lys?His?Pro?Pro?Val?Gln?Leu?Ala

20 25 30

Phe?Gln?Glu?Ile?Gly?Val?Asp?Arg?Ala?Glu?Glu?Val?Leu?Phe?Ser?Ala

35 40 45

Gly?Thr?Phe?Val?Arg?Leu?Glu?Phe?Lys?Leu?Gln?Gln?Thr?Asn?Cys?Pro

50 55 60

Lys?Lys?Asp?Trp?Lys?Lys?Pro?Glu?Cys?Thr?Ile?Lys?Pro?Asn?Gly?Arg

65 70 75 80

Arg?Arg?Lys?Cys?Leu?Ala?Cys?Ile?Lys?Met?Asp?Pro?Lys?Gly?Lys?Ile

85 90 95

Leu?Gly?Arg?Ile?Val?His?Cys?Pro?Ile?Leu?Lys?Gln?Gly?Pro?Gln?Asp

100 105 110

Pro?Gln?Glu?Leu?Gln?Cys?Ile?Lys?Ile?Ala?Gln?Ala?Gly?Glu?Asp?Pro

115 120 125

His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe?Ser?Arg?Ala?Leu?Arg

130 135 140

Thr?Lys

145

<210>36

<211>162

<212>PRT

<213>Mus?sp.

<400>36

Met?Lys?Cys?Leu?Leu?Ile?Ser?Leu?Ala?Leu?Trp?Leu?Gly?Thr?Arg?Gly

1 5 10 15

Thr?Val?Gly?Thr?Glu?Pro?Glu?Leu?Ser?Glu?Thr?Gln?Arg?Arg?Ser?Leu

20 25 30

Gln?Val?Ala?Leu?Glu?Glu?Phe?His?Lys?His?Pro?Pro?Val?Gln?Leu?Ala

35 40 45

Phe?Gln?Glu?Ile?Gly?Val?Asp?Arg?Ala?Glu?Glu?Val?Leu?Phe?Ser?Ala

50 55 60

Gly?Thr?Phe?Val?Arg?Leu?Glu?Phe?Lys?Leu?Gln?Gln?Thr?Asn?Cys?Pro

65 70 75 80

Lys?Lys?Asp?Trp?Lys?Lys?Pro?Glu?Cys?Thr?Ile?Lys?Pro?Asn?Gly?Arg

85 90 95

Arg?Arg?Lys?Cys?Leu?Ala?Cys?Ile?Lys?Met?Asp?Pro?Lys?Gly?Lys?Ile

100 105 110

Leu?Gly?Arg?Ile?Val?His?Cys?Pro?Ile?Leu?Lys?Gln?Gly?Pro?Gln?Asp

115 120 125

Pro?Gln?Glu?Leu?Gln?Cys?Ile?Lys?Ile?Ala?Gln?Ala?Gly?Glu?Asp?Pro

130 135 140

His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe?Ala?Phe?Ser?Arg?Ala?Leu?Arg

145 150 155 160

Thr?Lys

<210>37

<211>19

<212>PRT

<213>Mus?sp.

<400>37

Ala?Gln?Ala?Gly?Glu?Asp?Pro?His?Gly?Tyr?Phe?Leu?Pro?Gly?Gln?Phe

1 5 10 15

Ala?Phe?Ser

<210>38

<211>19

<212>PRT

<213>Homo?sapiens

<400>38

Gln?Arg?Ala?Gly?Glu?Asp?Pro?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe

1 5 10 15

Ala?Phe?Ser

<210>39

<211>15

<212>PRT

<213>Artificial

<220>

<223>Scrambled?C15

<400>39

Gly?Leu?Phe?His?Asp?Gln?Ala?Gly?Pro?Pro?Ala?Gly?Tyr?Glu?Phe

1 5 10 15

<210>40

<211>15

<212>PRT

<213>Artificial

<220>

<223>Mutant?C15

<400>40

Ala?Gly?Glu?Asp?Pro?His?Gly?Tyr?Ala?Leu?Pro?Gly?Gln?Ala?Ala

1 5 10 15

<210>41

<211>6

<212>PRT

<213>Mus?sp.

<400>41

Arg?Ala?Leu?Arg?Thr?Lys

1 5

<210>42

<211>8

<212>PRT

<213>Mus?sp.

<400>42

Phe?Ser?Arg?Ala?Leu?Arg?Thr?Lys

1 5

<210>43

<211>13

<212>PRT

<213>Artificial

<220>

<223>Modified?C13h

<400>43

Phe?His?Ser?Phe?Tyr?Phe?Pro?Gly?Gln?Phe?Ala?Phe?Ser

l 5 10

<210>44

<211>19

<212>PRT

<213>Mus?sp.

<400>44

Glu?Leu?Ser?Glu?Thr?Gln?Arg?Arg?Ser?Leu?Gln?Val?Ala?Leu?Glu?Glu

1 5 10 15

Phe?His?Lys

<210>45

<211>20

<212>PRT

<213>Mus?sp.

<400>45

Lys?Pro?Glu?Cys?Thr?Ile?Lys?Pro?Asn?Gly?Arg?Arg?Arg?Lys?Cys?Leu

1 5 10 15

Ala?Cys?Ile?Lys

20

Claims (28)

1. derived from one or more peptides of the proteic C-end of Kai Morui, or their analog or derivant are used for the treatment of application in the medicine of inflammation in preparation.

2. derived from one or more peptides of the proteic C-end of Kai Morui, or their analog or derivant are used for the treatment of application in the medicine of endotoxin shock in preparation.

3. derived from one or more peptides of the proteic C-end of Kai Morui, or their analog or derivant are used for reducing the application of the medicine of one or more inflammatory mediator levels in preparation.

4. a treatment, prevent or improve the method for the intravital inflammation of experimenter, comprise giving described experimenter one or more peptide or their analog or derivants derived from the proteic C-end of Kai Morui.

5. a treatment, prevent or improve the method for the intravital endotoxin shock of experimenter, comprise giving described experimenter one or more peptide or their analog or derivants derived from the proteic C-end of Kai Morui.

6. a method that reduces the level of intravital one or more inflammatory mediators of experimenter comprises giving described experimenter one or more peptide or their analog or derivants derived from the proteic C-end of Kai Morui.

7. application according to claim 3 or the described method of claim 6, wherein, described one or more inflammatory mediators are selected from the group that comprises TNF α, IL-1 α, IL-1 β, IL-6, IL-12, G-CSF, MCP-2 (CCL8), GRO α (CXCL1), GRO β (CXCL2), IL-8 (CXCL8), TECK (CCL25), MCP-1 (CCL2), interferon gamma and Rantes (CCL5).

8. derived from one or more peptides of the proteic C-end of Kai Morui, or their analog or derivant are used for handling the application of the medicine of wound in preparation.

9. a treatment, prevent or improve the method for experimenter's wound, comprise giving described experimenter one or more peptide or their analog or derivants derived from the proteic C-end of Kai Morui.

10. according to claim 1,2,3,7 or 8 described application, wherein, described medicine is used for the treatment of and/or prevention and/or cosmetic applications, perhaps according to claim 4,5,6,7 or 8 described methods, wherein, described treatment/be treated to curative and/or preventative and/or beauty treatment usefulness.

11. according to each described application or method in the aforementioned claim, wherein, described peptide arrives between about 30 aminoacid about 5.

12. according to each described application or method in the aforementioned claim, wherein, described peptide comprise derived from the proteic C-end of Kai Morui about 5 to about 30 aminoacid, or their analog or derivant.

13. according to each described application or method in the aforementioned claim, wherein, described peptide and the proteic naturally occurring C-end of described Kai Morui derived from the proteic C-end of Kai Morui has at least 30% or higher homogeneity.

14. according to each described application or method in the aforementioned claim, wherein, described peptide with according to the Kai Morui of Seq ID no:31 (human sequence) and Seq ID no:34 (mice sequence) proteic last 5 extremely last 30 aminoacid have at least 30% sequence homogeneity.

15. according to each described application or method in the aforementioned claim, wherein, described anti-inflammatory activity and/or the active and/or described inflammatory mediator level of described antiendotoxin shock at least about 50% with peptide of comprising naturally occurring sequence of described analog or derivant reduces active.

16. according to each described application or method in the aforementioned claim, wherein, one or more in the described peptide have Seq ID No:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 and 30 sequence or are their analog or derivant; Or

According to each described application or method in the aforementioned claim, wherein, one or more in the described peptide have the sequence of Seq ID No:37 and 38 or are their analog or derivant.

17. application according to claim 16 or method, wherein, one or more peptides among described peptide and the Seq IDNo:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 have at least 30% sequence homogeneity; Or application according to claim 16 or method, wherein, one or more peptides in described peptide and SeqID No:37 or 38 have at least 30% sequence homogeneity.

18. according to each described application or method in the claim 1 to 15, wherein, described peptide analogues or derivant comprise the small molecule mimetics derived from the peptide of the proteic C-end of Kai Morui.

19. according to each described application or method in the aforementioned claim, wherein, described peptide or their analog or derivant are intended to the dosed administration to about 1mg/kg with about 10pg/kg.

20. application according to claim 19 or method, wherein, described peptide or their analog or derivant are intended to the dosed administration to about 100ng/kg with about 10pg/kg.

21. with one or more medical apparatus derived from the peptide of the proteic C-end of Kai Morui or their analog or derivant dipping.

22. with one or more wound dressing or binders derived from the peptide of the proteic C-end of Kai Morui or their analog or derivant dipping.

23. a pharmaceutical composition, comprise one or more be selected from comprise have Seq ID No:1, peptide and medicinal diluent, carrier or excipient in the group that the peptide of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 and 30 sequence and their analog or derivant are formed; Or

A kind of pharmaceutical composition comprises one or more and is selected from peptide in the group that the peptide that comprises the sequence with Seq IDNo:37 and 38 and their analog or derivant form and medicinal diluent, carrier or excipient.

24. compositions according to claim 23 treats and/or prevents and/or the application that treats and/or prevents and/or be used for reducing the level of one or more inflammatory mediators of endotoxin shock inflammation.

25. the application of compositions according to claim 23 in handling wound.

26. have peptide or their analog or the derivant of Seq ID No:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 sequence; The peptide or their analog or the derivant that perhaps have sequence Seq ID No:37 or 38.

27. a peptide has at least 50% sequence homogeneity with the peptide with Seq ID No:1, sequence of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30; Or

A kind of peptide has at least 50% sequence homogeneity with the peptide of the sequence with Seq ID No:37 or 38.

28. one or more are derived from the peptide of the proteic C-end of Kai Morui, or their analog or derivant, treating and/or preventing and/or the level that treats and/or prevents and/or be used to reduce one or more inflammatory mediators such as cytokine and chemotactic factor of endotoxin shock of inflammation; Or be used for the application of the processing of wound.

Images